Compositions and methods that modulate vitamin d and bone mineral content in a companion animal

ABSTRACT

Bone mineral content, 25(OH) vitamin D3, or 1,25(OH) 2  vitamin D3 in a companion animal can be improved by adjusting the diet of the animal to increase the amount of a compound which positively or negatively modulates the bone mineral content, 25(OH) vitamin D3, or 1,25(OH) 2  vitamin D3 or adjusting the diet of the animal to decrease the amount of a compound which positively or negatively modulates the bone mineral content, 25(OH) vitamin D3, or 1,25(OH) 2  vitamin D3.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/839,333 filed Dec. 12, 2017, which claims priority to U.S. Provisional Application Ser. No. 62/434,556 filed Dec. 15, 2016, the disclosures of which are incorporated in their entireties herein by this reference.

BACKGROUND

Bone mineral content (BMC), also termed bone mineral density or bone density, is the amount of minerals present in bone (usually expressed as mineral/cm² bone). BMC is a common marker for diseases such as osteopenia and osteoporosis and can be used to predict the probability of bone fractures. In addition, low BMC may also be indicative of cancer, Cushing's syndrome, ankylosing spondylitis and vitamin D deficiency. Vitamin D has been associated with many other health and wellness areas as well. These include osteoporosis, rickets, osteomalacia, cancer, cognition, immune (autoimmune and infection), among others.

SUMMARY

The present disclosure relates generally to pet food compositions; methods of minimizing costs associated with production of a pet food; methods of enhancing nutritional benefit of a pet food; and methods for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal. Specifically, the present disclosure relates to metabolites for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal.

The present inventors have developed a predictive model of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 by identifying metabolite compounds which correlate to bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. A very controlled study was employed to minimize other external factors by using multiple canines all fed the same diet. A validation model was then developed by feeding different levels of the identified compounds (via a dietary change) to a group of canines and measuring changes in the corresponding health parameter (i.e., one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3).

Accordingly, in a general embodiment, the present disclosure provides a pet food composition comprising: protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal. In one embodiment, the pet food composition can provide at least a 5% increase or at least a 5% decrease in the amount of at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. In one aspect, the pet food can provide an increase. In another aspect, the pet food can comprise a decrease.

The present disclosure also provides methods of minimizing costs associated with production of a pet food; methods of enhancing nutritional benefit of a pet food; methods for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal; and methods for measuring a change in the amount of at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal.

An advantage of one or more embodiments provided by the present disclosure is to improve bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal by adjusting the diet of the animal to increase the amount of a compound which positively modulates bone mineral content, 25(OH) vitamin D3, or 1,25 (OH)₂ vitamin D3 or to decrease the amount of a compound which negatively modulates bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3.

Another advantage of one or more embodiments provided by the present disclosure is to minimize pet food production costs by utilizing ingredients which are appropriately high or low in the identified compounds or precursors thereof, as generally illustrated by the non-limiting examples discussed herein.

Additional features and advantages are described herein and will be apparent from the following Detailed Description.

DETAILED DESCRIPTION Definitions

As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” or “the composition” includes two or more compositions. The term “and/or” used in the context of “X and/or Y” should be interpreted as “X,” or “Y,” or “X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative, and are not exclusive or comprehensive.

As used herein, “about” is understood to refer to numbers in a range of numerals, for example the range of −10% to +10% of the referenced number, within −5% to +5% of the referenced number, or in one aspect, within −1% to +1% of the referenced number, and in a specific aspect, within −0.1% to +0.1% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All percentages expressed herein are by weight of the total weight of the food composition unless expressed otherwise. When reference is made to the pH, values correspond to pH measured at 25° C. with standard equipment. An “amount” can be the total amount of the referenced component per serving of the composition or per distinct unit of the composition and/or can be the weight percentage of the referenced component by dry weight. Moreover, an “amount” includes zero; for example, the recitation of an amount of a compound does not necessarily mean that the compound is present, unless followed by a range that excludes zero.

The terms “food,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an animal, including a human, and provides at least one nutrient to the animal. Further in this regard, these terms mean that the product or composition is in a form ready for consumption and is not merely an intermediate from which a consumable product or composition is made, although other food compositions can be added in some embodiments. The term “pet food” means any food composition intended to be consumed by a pet. The term “pet” means any animal which could benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal.

The term “companion animal” means a dog or a cat. In an embodiment, the compositions and methods disclosed herein involve a senior dog or a senior cat. Dogs are considered senior in the last 25% of their lives. The life span of a dog depends on its size and/or its breed, but for the present disclosure a senior dog is a dog that is at least 5 years of age (e.g., at least 6 years of age, at least 7 years of age, or at least 8 years of age). The life span of a cat also depends on its size and/or its breed, but for the present disclosure a senior cat is a cat that is at least 7 years of age (e.g., at least 8 years of age, at least 9 years of age, or at least 10 years of age).

As used herein, “comparable companion animal” refers to a healthy animal of the same gender, breed, and age as the companion animal.

As used herein, “metabolite” refers to a compound having biological activity in a companion animal that is an intermediate or product of metabolism, and includes precursors thereof. As used herein, “precursor” refers to any compound that metabolizes to a metabolite during metabolism in a companion animal. For example, if the specific metabolite is cysteine, “the metabolite” comprises a cysteine precursor (e.g., methionine).

The term “effective amount” means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In one embodiment, the present metabolite or combination of metabolites can be present in an effective amount for modulating bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal.

The dosages expressed herein are in milligrams per kilogram of body weight per day (mg/kg/day) unless expressed otherwise.

The term “long-term administration” means periods of repeated administration or consumption in excess of one month. Periods of longer than two, three, or four months can be used for certain embodiments. Also, more extended periods can be used that include longer than 5, 6, 7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year can also be used. Longer term use extending over 1, 2, 3, or more years are included in the invention. For certain aging animals, the animal will continue consuming on a regular basis for the remainder of its life. This can also be referred to as consumption for “extended” periods.

The term “regular basis” or “regular administration” means at least monthly dosing with the compositions or consumption of the compositions, and in one aspect, means at least weekly dosing. More frequent dosing or consumption, such as twice or three times weekly, can be performed in certain embodiments. Still, in other embodiments, regimens can be used that comprise at least once daily consumption. The skilled artisan will appreciate that the blood level of a compound or certain metabolites of that compound or which result after the consumption of that compound, may be a useful tool for assessing or determining dosing frequency. For example, for determining feeding amounts for pet food compositions comprising a certain metabolite, the blood concentration of that metabolite, may provide useful information. A frequency, regardless of whether expressly exemplified herein, that allows maintenance of a desired blood level of the measured compound, such as a metabolite, within acceptable ranges can be useful herein. The skilled artisan will appreciate that feeding amounts will be a function of the composition that is being consumed or administered as well as the animal consuming the food, and some food compositions may require more or less frequent administration to maintain a desired blood level of the measured compound (e.g., a metabolite).

The relative terms “improve,” “increase,” “enhance,” “decrease” and the like refer to the effects of the composition disclosed herein (a composition comprising a metabolites) relative to a composition having a lower amount or lacking such metabolites, but otherwise identical.

A “blended” composition merely has at least two components having at least one different characteristic relative to each other. In one aspect, moisture content and water activity can be different in the context of the present disclosure. In this regard, description of a composition as “blended” does not imply that the blended composition has been subjected to processing sometimes referenced as “blending,” namely mixing components so that they are indistinguishable from each other, and, in one aspect, such processing is avoided when mixing one component with the other components to form a blended composition (e.g., mixing a dry component with a wet or semi-moist component). Further in this regard, in a blended composition each of the at least two components having at least one different characteristic relative to each other can retain their distinct identity and appearance.

“Wet food” means a pet food having a moisture content from about 50% to about 90%, and in one aspect, from about 70% to about 90%. “Dry food” means a pet food having a moisture content less than about 20%, and in one aspect, less than about 15%, and in a specific aspect, less than about 10%. “Semi-moist food” means a pet food having a moisture content from about 20% to about 50%, and in one aspect, from about 25% to about 35%.

“Kibbles” is used synonymously with “chunks” herein and both terms mean pieces of dry or semi-moist pet food which can have a pellet shape or any other shape and can be made by slicing a food composition into separate pieces. Non-limiting examples of kibbles include particulates; pellets; pieces of pet food, dehydrated meat, meat analog, vegetables, and combinations thereof; and pet snacks, such as meat or vegetable jerky, rawhide, and biscuits. A “meat analog” is a meat emulsion product that resembles pieces of natural meat in appearance, texture, and physical structure.

The term “dietary supplement” means a product that is intended to be ingested in addition to the normal animal diet. Dietary supplements may be in any form, e.g., solid, liquid, gel, tablets, capsules, powder, and the like. In one aspect, they can be provided in convenient dosage forms. In some embodiments, they can be provided in bulk consumer packages such as bulk powders, liquids, gels, or oils. In other embodiments, supplements can be provided in bulk quantities to be included in other food items such as snacks, treats, supplement bars, beverages and the like.

The compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the components identified. Similarly, the methods disclosed herein may lack any step that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of” and “consisting of” the steps identified. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly and directly stated otherwise.

The term “bone mineral content” or “bone density” refers to the amount of bone mineral in bone tissue and, unless otherwise stated, is measured as mass of mineral per volume of bone (g/cm²).

The term “25(OH) vitamin D3” or “calcifediol” or “25-hydroxycholecalciferol” refers to a prehormone that is produced in the liver by hydroxylation of vitamin D3 (cholecalciferol) by the enzyme cholecalciferol 25-hydroxylase and has the following structure:

The term “1,25(OH)₂ vitamin D3” or “calcitriol” or “1,25-dihydroxycholecalciferol” refers to the hormonally active metabolite of vitamin D with three hydroxyl groups (which can be abbreviated 1,25-(OH)2D3 or 1,25(OH)2D) and has the following structure:

The present discussion of embodiments, aspects, examples, etc. are independent in that they can apply to all methods and compositions. For example, a metabolite used in a pet food composition can also be used in the method of modulating or a method of minimizing costs associated with making such a pet food, and vice versa.

Embodiments

In an aspect of the present disclosure, a pet food composition can comprise protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal. In one aspect, the pet food composition can include at least 6 distinct metabolites including at least 2 distinct metabolites for modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 in a companion animal. In one embodiment, the pet food composition can provide an increase in at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

In another aspect of the present disclosure, a method of modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal is provided. The method comprises administering to the companion animal a pet food composition comprising protein, carbohydrates, fat, fiber, and a metabolite for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. In one aspect, the companion animal can be a senior dog or a senior cat.

In still another aspect of the present disclosure, a method of measuring a change in the amount of at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal can comprise obtaining a serum or plasma sample of the companion animal, measuring concentrations of at least three distinct metabolites from the serum sample that modulate at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3, and determining that the bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 has changed if, after comparing the metabolite concentrations to average metabolite concentrations of each metabolite from comparable companion animals, the metabolite concentrations are different than the average metabolite concentrations.

Yet another aspect of the present disclosure is a method of minimizing costs associated with production of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. A further aspect of the present disclosure is a method of enhancing nutritional benefit of a pet food having a first formulation designed for consumption by a companion animal, such as a senior dog or a senior cat. These methods comprise adjusting the first formulation of the pet food to be a second formulation. At least one of the first and second formulations comprises a metabolite for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. The adjusting comprises changing an amount of the metabolite in the first formulation to a different amount in the second formulation.

“Minimizing” costs means that the costs associated with making the second formulation are less than the costs associated with making the first formulation, for example on a per serving basis, per unit weight, per unit volume, per total energy, and the like. “Enhanced” nutritional benefit means that the nutritional benefit of the second formulation is greater than the nutritional benefit of the first formulation.

As discussed herein, the pet food compositions and methods can contain a metabolite or multiple metabolites for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. In various aspects, the compositions and methods can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 metabolites for any one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3, or at least two of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3, or for each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3. As such, in one embodiment, the composition can comprise at least 9 distinct metabolites, including at least 3 distinct metabolites for individually modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 in a companion animal. In another embodiment, the composition can comprise at least 12 distinct metabolites, including at least 4 distinct metabolites for individually modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 in a companion animal. Additionally, in one embodiment, the composition can comprise at least four distinct metabolites that modulate at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. Further, in one embodiment, the composition can comprise at least five distinct metabolites that modulate at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. Still, in another embodiment, the method can modulate at least two members selected from the group consisting of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 and the composition can comprise at least six distinct metabolites, three distinct metabolites for modulating each of the at least two members. Yet, in another embodiment, the method can modulate each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 and the composition can comprise at least nine distinct metabolites, three distinct metabolites for modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3. As previously noted, the metabolite/health parameter relationships can be used for both method and composition embodiments.

In some embodiments, the metabolite negatively modulates the health parameter, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. In other embodiments, the metabolite negatively modulates the health parameter, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In some embodiments, the metabolite positively modulates the health parameter, and the changing of the amount of the metabolite comprises increasing the amount of the metabolite. In other embodiments, the metabolite positively modulates the health parameter, and the changing of the amount of the metabolite comprises decreasing the amount of the metabolite. These are not mutually exclusive embodiments; a particular embodiment can comprise decreasing the amount of a metabolite that negatively modulates a first health parameter and increasing the amount of a metabolite that positively modulates a second health parameter, and the first and second parameters can be the same or different health parameters (e.g., one or more of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3).

Decreasing the amount of the metabolite can comprise decreasing the amount of the metabolite directly and/or decreasing the amount of an ingredient which comprises the metabolite. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

Increasing the amount of the metabolite can comprise increasing the amount of the metabolite directly and/or increasing the amount of an ingredient which comprises the metabolite. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

Generally, the methods and compositions described herein can provide an increase or a decrease in at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. In some embodiment, the increase or decrease can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50%. In one aspect, the compositions and methods can provide an increase. In another aspect, the compositions and methods can provide a decrease. As such, the present modulation can treat osteoporosis, osteopenia, rickets, osteomalacia, cognitive disorders, immune deficiency, autoimmune diseases (including but not limited to, multiple sclerosis, rheumatoid arthritis, Crohn's disease), heart diseases, diabetes, or cancer (including but not limited to lymphocytic leukemia, melanoma, colorectal, kidney, bladder). Additionally, the present modulation can increase or decrease bone growth, improve the immune system, increase skin health, increase hair health, or increase bone health. Further, the present modulation, and resulting increase or decrease, can affect cellular mechanisms associated with health and disease areas. These mechanisms can include cell proliferation, differentiation, apoptosis, cell migration, DNA repair, angiogenesis and immune surveillance.

In one embodiment, the increase or decrease can be for at least two of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3. In another embodiment, the increase or decrease can be for each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3.

The cost of the pet food can be minimized and/or the nutritional benefit of the pet food can be enhanced by utilizing ingredients which are appropriately high in compounds which positively modulate at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 and/or appropriately low in compounds which negatively modulate at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3.

Ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

In each of these compositions and methods, the pet food composition can be a wet food, a semi-moist food or a dry food. In an embodiment, the pet food composition is one or more components of a blended composition. In some embodiments, the pet food composition is a kibble, and in some embodiments, the pet food composition is a meat analog. Additionally, in another embodiment, the present composition for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 can be a dietary supplement comprising the metabolites described herein. Further, a method of modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 can include administering to the companion animal the dietary supplement.

Such pet food compositions can be administered to the companion animal in amounts ranging from about 3 g of pet food per 1 lb body weight to about 16 g of pet food per 1 lb body weight of the companion animal. Additionally, the metabolites can be present in amounts from about 0.01 weight % to about 10 weight % of the food composition. In one aspect, the metabolites can be present in concentrations of about 0.01 to about 1,000 mg/kg of food. In another aspect, the metabolites can be present in concentrations from about 1 IU to about 500,000 IU per kilogram of food. In one embodiment, the pet food composition can be administered to the companion animal in amounts sufficient to maintain the health and/or body weight of the animal. In one aspect, the administration can be regular administration.

As noted above and detailed later in this application, the present inventors identified metabolite compounds which correlate to bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3. Thus, the metabolite in the pet food composition can be one of these compounds. Nevertheless, the metabolite can be any metabolite for modulating at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in a companion animal, even if the metabolite is not explicitly disclosed herein. For example, the metabolite can be a compound identified using the methods disclosed herein but not itself explicitly disclosed herein. Furthermore, the metabolite can be a compound identified using a method not disclosed herein if the compound is reliably correlated to at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3.

As a non-limiting example, the metabolite can modulate bone mineral content and can be selected from the group consisting of creatinine, X-14625, gamma-glutamylleucine, ethanolamine, histidine, 4-acetylphenyl sulfate, phosphoethanolamine (PE), X-14352, pseudouridine, myo-inositol, X-15375, X-17299, 1-methylguanosine, xylitol, X-11787, pyroglutamine, and mixtures thereof. In one embodiment, the metabolite can modulate bone mineral content and can be selected from the group consisting of creatinine, gamma-glutamylleucine, ethanolamine, histidine, 4-acetylphenyl sulfate, phosphoethanolamine (PE), pseudouridine, 1-methylguanosine, xylitol, pyroglutamine, and mixtures thereof.

As another non-limiting example, the metabolite can modulate 25(OH) vitamin D3 and can be selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), X-17612, mannose, X-17147, anserine, X-17010, cytidine, prolylhydroxyproline, arabonate, 3-hydroxybutyrate (BHBA), docosapentaenoate (DPA; 22:5n3), adrenate (22:4n6), X-12195, N-acetylalanine, dihomolinoleate (20:2n6), stearate (18:0), palmitate (16:0), arabitol, taurolithocholate, glutamate, 17-methylstearate, docosapentaenoate (n6 DPA; 22:5n6), sphingomyelin, glycerate, oleate (18:1n9), X-11378, and mixtures thereof. In one embodiment, the metabolite can modulate 25(OH) vitamin D3 and can be selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), mannose, anserine, cytidine, prolylhydroxyproline, arabonate, 3-hydroxybutyrate (BHBA), docosapentaenoate (DPA; 22:5n3), adrenate (22:4n6), N-acetylalanine, dihomolinoleate (20:2n6), stearate (18:0), palmitate (16:0), arabitol, taurolithocholate, glutamate, 17-methylstearate, docosapentaenoate (n6 DPA; 22:5n6), sphingomyelin, glycerate, oleate (18:1n9), and mixtures thereof.

As yet another non-limiting example, the metabolite can modulate 1,25(OH)2 vitamin D3 and can be selected from the group consisting of arachidonate (20:4n6), X-11378, pantothenate (Vitamin B5), mannose, isobutyrylcarnitine (C4), sphingomyelin, 3-hydroxybutyrate (BHBA), prolylhydroxyproline, glycerate, dihomolinolenate (20:3n3 or 3n6), X-17010, adrenate (22:4n6), alpha-tocopherol, caproate (6:0), octanoylcarnitine (C8), and mixtures thereof. In one embodiment, the metabolite can modulate 1,25(OH)₂ vitamin D3 and can be selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), mannose, isobutyrylcarnitine (C4), sphingomyelin, 3-hydroxybutyrate (BHBA), prolylhydroxyproline, glycerate, dihomolinolenate (20:3n3 or 3n6), adrenate (22:4n6), alpha-tocopherol, caproate (6:0), octanoylcarnitine (C8), and mixtures thereof.

In yet another aspect of the present disclosure, a method enhances nutritional benefit of a pet food having a first formulation designed for consumption by companion animals, and the method comprises administering the pet food having the first formulation to a first companion animal. The method further comprises measuring in a sample of body fluid from the companion animal (e.g., plasma) an amount of a surrogate marker comprising a metabolite that modulates at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. The method further comprises adjusting the first formulation of the pet food to be a second formulation having a difference from the first formulation selected from the group consisting of (i) an ingredient is present in the second formulation and is absent in the first formulation, (ii) an ingredient is absent in the second formulation and is present in the first formulation, (iii) an ingredient is present in the first and second formulations but in a different amount, and (iv) combinations thereof. The adjusting is based at least partially on the amount of the surrogate marker measured in the previous step.

The adjusting can comprise directly decreasing the amount of a metabolite negatively modulating a specific health parameter and/or decreasing the amount of an ingredient which comprises a metabolite negatively modulating a specific health parameter. In some embodiments, decreasing the amount of the metabolite can comprise decreasing the amount of a precursor of the metabolite directly and/or decreasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, less of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

The adjusting can comprise increasing the amount of the metabolite can comprise directly increasing the amount of a metabolite positively modulating a specific health parameter and/or increasing the amount of an ingredient which comprises a metabolite positively modulating a specific health parameter. In some embodiments, increasing the amount of the metabolite can comprise increasing the amount of a precursor of the metabolite directly and/or increasing the amount of an ingredient which comprises a precursor of the metabolite. For example, the second formulation can contain, relative to the first formulation, more of an ingredient having a high amount of the metabolite or precursor thereof (e.g., an ingredient having an amount of the metabolite or precursor thereof that is higher than in one or more of the other ingredients).

As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

The method further comprises producing the pet food in the second formulation. In an embodiment, the method comprises administering the pet food having the second formulation to a second companion animal.

This method can be used to provide customized nutrition for a specific companion animal. For example, the first and second companion animal can be the same specific companion animal such that the animal who is administered the pet food having the first formulation has one or more of their specific health parameters of the first formulation assessed. Then this same animal is provided with the resultant second formulation which will more effectively provide at least one of bone mineral content, 25(OH) vitamin D3, or 1,25 (OH)₂ vitamin D3 to the companion animal. Consequently, a pet owner can compensate for their pet's age-induced changes in one or more of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3.

Alternatively or additionally, this method can be used to provide customized nutrition for companion animals who share one or more of a gender, an approximate age, an approximate size (e.g., body weight, height, and/or length) or a breed. For example, the second companion animal can be a different specific animal than the first companion animal but has a characteristic selected from the group consisting of (i) about the same age as the first companion animal, (ii) about the same size as the first companion animal, (iii) the same breed as the first companion animal, (iv) the same gender as the first companion animal, and (iv) combinations thereof. In one embodiment, the second companion animal can be one of a plurality of companion animals who each share the characteristic with the first companion animal. The method can comprise administering the pet food having the second formulation to the plurality of companion animals. In an embodiment, at least a portion of the plurality of companion animals is located remotely relative to the first companion animal.

The pet food compositions disclosed herein can be any food formulated for consumption by a pet such as a companion animal. In an embodiment, the pet food composition provides complete nutrition as defined by the Association of American Feed Control Officials (AAFCO) and which depends on the type of animal for which the composition is intended (e.g., dog or cat).

The pet food composition can comprise meat, such as emulsified meat. Examples of suitable meat include poultry, beef, pork, lamb and fish, especially those types of meats suitable for pets. The meat can include any additional parts of an animal including offal. Some or all of the meat can be provided as one or more meat meals, namely meat that has been dried and ground to form substantially uniform-sized particles and as defined by AAFCO. Additionally or alternatively, vegetable protein can be used, such as pea protein, corn protein (e.g., ground corn or corn gluten), wheat protein (e.g., ground wheat or wheat gluten), soy protein (e.g., soybean meal, soy concentrate, or soy isolate), rice protein (e.g., ground rice or rice gluten) and the like.

The pet food compositions disclosed herein can comprise vegetable oil, a flavorant, a colorant and water. Suitable vegetable oils include soybean oil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil, safflower oil, and the like. Examples of suitable flavorants include yeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, pork), flavor extracts or blends (e.g., grilled beef), animal digests, and the like. Suitable colorants include FD&C colors, such as blue no. 1, blue no. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6, and the like; natural colors, such as caramel coloring, annatto, chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene, elderberry juice, pandan, butterfly pea and the like; titanium dioxide; and any suitable food colorant known to the skilled artisan.

The pet food compositions disclosed herein can optionally include additional ingredients, such as other grains and/or other starches additionally or alternatively to flour, amino acids, fibers, sugars, animal oils, aromas, other oils additionally or alternatively to vegetable oil, humectants, preservatives, polyols, salts, oral care ingredients, antioxidants, vitamins, minerals, probiotic microorganisms, bioactive molecules or combinations thereof.

Suitable starches include a grain such as corn, rice, wheat, barley, oats, soy and the like, and mixtures of these grains, and can be included at least partially in any flour. Suitable humectants include salt, sugars, propylene glycol and polyhydric glycols such as glycerin and sorbitol, and the like. Suitable oral care ingredients include alfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate, phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodium pyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves, parsley, ginger and the like. Examples of suitable antioxidants include butylated hydroxyanisole (“BHA”) and butylated hydroxytoluene (“BHT”), vitamin E (tocopherols), and the like.

Non-limiting examples of vitamins that can be used include Vitamins A, B-complex (such as B-1, B-2, B-6 and B-12), C, D, E and K, niacin and acid vitamins such as pantothenic acid and folic acid and biotin. Non-limiting examples of suitable minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium, boron and the like.

Non-limiting examples of suitable preservatives include potassium sorbate, sorbic acid, sodium methyl para-hydroxybenzoate, calcium propionate, propionic acid, and combinations thereof.

Specific amounts for each additional ingredient in the pet food compositions disclosed herein will depend on a variety of factors such as the ingredient included in the first edible material and any second edible material; the species of animal; the animal's age, body weight, general health, sex, and diet; the animal's consumption rate; the purpose for which the food product is administered to the animal; and the like. Therefore, the components and their amounts may vary widely.

For example, the amount of any of the above-noted ingredients can be decreased or increased based on the estimated effect on one or more of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 (e.g., an effect identified by one of the methods disclosed herein). In an embodiment, the amount of one or more of the above-noted ingredients can be increased if such ingredients comprise a metabolite that positively modulates one or more of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3. Additionally or alternatively, the amount of one or more of the above-noted ingredients can be decreased if such ingredients comprise a metabolite that negatively modulates one or more of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3.

As noted above, ingredients comprising the metabolite (e.g., a precursor of the metabolite) and optionally amounts of the metabolite in the ingredient can be identified by analysis of the ingredient, for example using a separation technique, such as gas chromatography or liquid chromatography, and then mass spectrometry.

EXAMPLES

The following non-limiting examples are illustrative of embodiments of the present disclosure.

Methods

Each of the examples was derived from the following study.

83 Canines were all fed Diet A for 5 weeks, followed by a 1 week transition period and then 15 were fed Diet B for 5 weeks as shown in the Table 1 below. Plasma samples were taken after overnight fasting using EDTA vacutainer tubes during the fifth week of feeding of each diet. After centrifugation, plasma was aliquoted into cryovials and frozen at −80° C. 25(OH) vitamin D3 and 1,25(OH)₂ vitamin D3 levels were determined according to commonly used assays. Bone density was measured using dual-energy x-ray absorptiometry (DEXA) according to manufacturer's directions.

TABLE 1 Moisture Moisture DM Protein Fat Ash Fiber CHO GE Basis % % % % % % % kcal/g Diet A As-Is 8.1 91.9 22.7 13.3 6.1 2.0 47.9 4.5 Dry 0 100 24.7 14.5 6.6 2.1 52.1 4.9 matter Diet B As-Is 76 24 9.1 10.5 1.8 0 2.6 1.7 Dry 0 100 38 43.7 7.5 0 10.8 6.9 Matter

Metabolomic analysis was carried out using the following methods by Metabolon Inc. Samples were extracted and split into equal parts for analysis on GC/MS and LC/MS/MS platforms. Proprietary software was used to match ions to an in-house library of standards for metabolite identification and for metabolite quantitation by peak area integration by Metabolon Inc. Mass and retention index are provided in the following tables such that each metabolite can be uniquely identified and individually distinguished.

At the time of analysis, samples were thawed and extracts prepared to remove protein, dislodge small molecules bound to protein or physically trapped in the precipitated protein matrix, and recover a wide range of chemically diverse metabolites. A separate aliquot of each experimental plasma sample was taken then pooled for the creation of “Client Matrix” (CMTRX) samples. These CMTRX samples were injected throughout the platform run and served as technical replicates, allowing variability in the quantitation of all consistently detected biochemicals to be determined and overall process variability and platform performance to be monitored. Extracts of all experimental and CMTRX samples were split for analysis on the GC/MS and LC/MS/MS platforms.

The CMTRX technical replicate samples were treated independently throughout the process as if they were client study samples. All process samples (CMTRX and Grob test mixtures of organic components used to assess GC column performance, process blanks, etc.) were spaced evenly among the injections for each day and all client samples were randomly distributed throughout each day's run. Data were collected over multiple platform run days and thus ‘block normalized’ by calculating the median values for each run-day block for each individual compound. This normalization minimizes any inter-day instrument gain or drift, but does not interfere with intra-day sample variability. Missing values (if any) were assumed to be below the level of detection for that biochemical with the instrumentation used and were imputed with the observed minimum for that particular biochemical.

A number of internal standards were added to each experimental and process standard sample just prior to injection into the mass spectrometers. A measure of the platform variability (7%) was determined by calculating the median relative standard deviation (RSD) for these internal standards. Because these standards are added to the samples immediately prior to injection into the instrument, this value reflects instrument variation. In addition, the median relative standard deviation (RSD) for the biochemicals that were consistently measured in the CMTRX represents the total variability within the process for the actual experimental samples and the variability in quantitation of the endogenous metabolites within these samples (12%). Results for the CMTRX and internal standards indicated that the platform produced data that met process specifications.

589 total metabolites were detected in plasma. This total corresponds to many biochemicals (401) that matched a named structure in the reference library (named compounds). The remaining biochemicals (188) represent distinct chemical entities (that is, they represent a single molecule of discrete molecular formula and structure), but they do not currently match a named biochemical in the reference library (unnamed/unknown compounds).

Example 1 (Bone Mineral Content)

Metabolite correlations with bone mineral content were determined based on plasma metabolomics (Table 2). This provided a predictive model of compounds which can influence bone mineral content either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 3), and noting changes in bone mineral content (Table 4), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those validated by the model are shown in Table 5.

TABLE 2 Metabolite correlations with bone mineral content. Correlations with a P value < 0.01 are reported. Corre- Correlation Retention ID lation P-Value Index Mass 5-oxoproline 0.6843 1.36E−12 744 128.2 phenylalanine 0.6410 8.78E−11 2056 166.1 creatinine 0.5922 4.64E−09 730 114.1 gamma- 0.5875 6.53E−09 2846 295.1 glutamylphenylalanine hydroxyproline 0.5846 8.07E−09 705 132.1 creatine −0.5462 1.11E−07 758 132.1 glutamine 0.5189 5.90E−07 684 147.2 X-14625 0.5162 6.89E−07 742 308.1 X-11334 0.5073 1.15E−06 982 259.1 urate 0.5029 1.47E−06 1928 441.2 gamma-glutamylleucine 0.5010 1.64E−06 2744 261.2 X-18487 0.4880 3.31E−06 1269.6 273.1 hydroquinone sulfate −0.4610 1.31E−05 1383 189 X-14314 0.4540 1.84E−05 2302 241.1 X-11441 0.4337 4.70E−05 3773 331.1 X-11843 0.4336 4.72E−05 2710 230.1 mannitol 0.4228 7.57E−05 1839 319.1 ethanolamine 0.4227 7.63E−05 1304 174.1 X-11530 0.4098 1.31E−04 4866 313.2 gamma-glutamylisoleucine 0.4085 1.39E−04 2644 261.2 X-12236 0.4066 1.51E−04 1321 245.1 tyrosine 0.4062 1.53E−04 1516 182.1 X-11442 0.4062 1.53E−04 3902 331.1 gamma-glutamylmethionine 0.4045 1.64E−04 1993 279.2 2′-deoxycytidine 0.3955 2.35E−04 1256 228 phenol sulfate −0.3939 2.51E−04 2150 173.1 X-12010 0.3889 3.05E−04 1707 203.1 bilirubin (E, E) 0.3862 3.38E−04 4625 585.2 X-12822 0.3853 3.52E−04 2786 389.1 X-09789 0.3818 4.01E−04 2613 153.1 3-(3-hydroxyphenyl) 0.3817 4.03E−04 2000 165.1 propionate dihomolinolenate −0.3767 4.87E−04 5600 305.4 (20:3n3 or 3n6) 2-hydroxyisobutyrate −0.3747 5.24E−04 1107.5 130.9 gamma-glutamylvaline 0.3731 5.56E−04 2040 247.2 N-acetyltyrosine −0.3713 5.94E−04 1677 222.2 histidine 0.3705 6.12E−04 757 154.1 4-acetylphenyl sulfate 0.3701 6.21E−04 2399 215 3-methoxytyrosine 0.3696 6.32E−04 1765 212.1 tryptophan 0.3691 6.43E−04 2445 205.1 phenylalanyltryptophan 0.3689 6.49E−04 3349 352.2 indolepropionate 0.3671 6.91E−04 1925 202.1 phosphoethanolamine (PE) 0.3642 7.69E−04 1577.3 299.1 ophthalmate 0.3608 8.67E−04 1457 290.1 gamma-glutamyltyrosine 0.3578 9.66E−04 2073 311.2 3-(4-hydroxyphenyl) 0.3482 0.001 1727.9 179.1 propionate N-acetylornithine 0.3440 0.002 875 175.2 X-14352 −0.3438 0.002 2635 247.2 3-phenylpropionate 0.3342 0.002 2830 149.1 (hydrocinnamate) X-13835 −0.3332 0.002 1014 169.1 cysteine-glutathione 0.3326 0.002 821 427.1 disulfide X-11378 −0.3292 0.003 5325 445.4 pseudouridine 0.3286 0.003 1104 243.1 methionine 0.3230 0.003 1252 150.1 myo-inositol 0.3208 0.003 1924.9 217 X-15461 0.3194 0.003 2125 160.1 X-15375 0.3190 0.003 2502 328 X-17299 0.3183 0.004 1265.9 229.2 gamma-glutamylalanine 0.3158 0.004 1108 219 1-methylguanosine 0.3156 0.004 1850 298.1 X-12156 0.3146 0.004 3238 233.1 X-11949 −0.3121 0.004 3830 220.1 xylitol 0.3120 0.004 1677.6 217 N6-carbamoyl- 0.3100 0.005 2656 413 threonyladenosine equol sulfate 0.3095 0.005 3625 321.2 2-hydroxyglutarate −0.3089 0.005 1576 247 5,6-dihydrouracil 0.3066 0.005 1505.2 243 X-11787 0.3058 0.005 1126 148.1 pyroglutamine 0.3043 0.005 764 129.2 kynurenine 0.3038 0.006 1902 209.1 indoleacetylglutamine 0.3027 0.006 2530 302.2 glycerate −0.2999 0.006 1360.7 189 X-16940 −0.2978 0.007 1694.1 204.9 X-18570 0.2962 0.007 3243.1 207 indoleacetate 0.2958 0.007 3760 176.1 3-methylhistidine −0.2953 0.007 911 168.1 trigonelline 0.2880 0.009 757 138.1 (N′-methylnicotinate) leucine 0.2865 0.009 1674 132.2 stachydrine 0.2852 0.009 860 144.1 indoleacrylate 0.2834 0.010 2529 186.1

TABLE 3 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound). ID A B 5-oxoproline 6.61 6.58 phenylalanine 0.75 0.64 creatinine 13.3 27.38 gamma-glutamylphenylalanine 14.27 1.36 hydroxyproline 5.01 4.88 creatine 1.98 10.6 glutamine 0.18 0.18 X-14625 2.66 2.74 X-11334 0.19 0.19 urate 1.75 1.29 gamma-glutamylleucine 0.55 0.69 X-18487 0.26 0.26 hydroquinone sulfate 0.03 0.03 X-14314 23.7 1.51 X-11441 0.36 0.36 X-11843 0.19 0.19 mannitol 12.51 1.72 ethanolamine 30.73 86.35 X-11530 0.21 0.21 gamma-glutamylisoleucine 0.93 0.53 X-12236 0.1 0.1 tyrosine 0.82 0.86 X-11442 0.28 0.28 gamma-glutamylmethionine 0.47 0.44 2′-deoxycytidine 0.5 0.5 phenol sulfate 0.07 0.07 X-12010 0.5 0.5 bilirubin (E, E) 0.32 0.32 X-12822 0.2 0.2 X-09789 5.54 0.2 3-(3-hydroxyphenyl)propionate 0.1 0.1 dihomolinolenate (20:3n3 or 3n6) 0.42 5.03 2-hydroxyisobutyrate 0.51 0.51 gamma-glutamylvaline 0.4 0.26 N-acetyltyrosine 0.82 0.32 histidine 0.31 0.45 4-acetylphenyl sulfate 0.4 0.56 3-methoxytyrosine 0.48 0.48 tryptophan 0.29 0.18 phenylalanyltryptophan 0.29 0.29 indolepropionate 0.07 0.07 phosphoethanolamine (PE) 1.68 5.87 ophthalmate 1.86 0.38 gamma-glutamyltyrosine 16.98 2.04 3-(4-hydroxyphenyl)propionate 14.94 0.38 N-acetylornithine 0.41 0.21 X-14352 0.69 0.46 3-phenylpropionate (hydrocinnamate) 18.68 0.13 X-13835 0.4 0.4 cysteine-glutathione disulfide 0.04 0.04 X-11378 0.05 0.11 pseudouridine 0.07 0.1 methionine 0.55 0.51 myo-inositol 7.85 12.2 X-15461 0.97 0.35 X-15375 0.41 0.97 X-17299 1.67 1.84 gamma-glutamylalanine 1.56 1.06 1-methylguanosine 0.52 3.87 X-12156 0.19 0.19 X-11949 0.28 0.28 xylitol 8.06 10.23 N6-carbamoylthreonyladenosine 0.57 0.57 equol sulfate 0.08 0.08 2-hydroxyglutarate 2.26 3.52 5,6-dihydrouracil 1.87 0.18 X-11787 0.04 0.06 pyroglutamine 1.16 1.69 kynurenine 0.05 0.04 indoleacetylglutamine 0.52 0.52 glycerate 3.38 9.94 X-16940 0.12 0.12 X-18570 0.08 0.08 indoleacetate 1.78 0.46 3-methylhistidine 0.26 0.76 trigonelline (N′-methylnicotinate) 12.47 1.65 leucine 0.89 0.66 stachydrine 3.62 0.78 indoleacrylate 0.06 0.06

TABLE 4 Bone mineral content in response to diet A and Diet B. Diet A Diet B Difference Parameter Mean StdDev Mean StdDev Mean StdErr p-value Bone 443.6 77.2 450.2 79.5 −6.6 2.0 0.006 Mineral Content

TABLE 5 Compounds validated by dietary change. ID Retention Index Mass creatinine 730 114.1 X-14625 742 308.1 gamma-glutamylleucine 2744 261.2 ethanolamine 1304 174.1 histidine 757 154.1 4-acetylphenyl sulfate 2399 215 phosphoethanolamine (PE) 1577.3 299.1 X-14352 2635 247.2 pseudouridine 1104 243.1 myo-inositol 1924.9 217 X-15375 2502 328 X-17299 1265.9 229.2 1-methylguanosine 1850 298.1 xylitol 1677.6 217 X-11787 1126 148.1 pyroglutamine 764 129.2

Example 2 (25(OH) Vitamin D3)

Metabolite correlations with 25(OH) vitamin D3 were determined based on plasma metabolomics (Table 6). This provided a predictive model of compounds which can influence 25(OH) vitamin D3 either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 7), and noting changes in 25(OH) vitamin D3 (Table 8), served as a validation model. The metabolite compositions of the two different diets were determined to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 9).

TABLE 6 Metabolite correlations with 25(OH) vitamin D3. Correlations with a P value < 0.01 are reported. Corre- Correlation Retention ID lation P-Value Index Mass 3-dehydrocarnitine 0.5377 1.61E−08 1020 160.2 X-16009 0.5183 6.36E−08 2191 222 arachidonate (20:4n6) 0.4975 2.52E−07 5525 303.4 pantothenate 0.4787 8.11E−07 2218 220.1 (Vitamin B5) X-17612 0.4742 1.06E−06 4298.5 567.4 benzoate 0.4584 2.64E−06 1291.5 179 1,5-anhydroglucitol −0.4429 6.23E−06 1788.7 217 (1,5-AG) X-11852 0.4269 1.44E−05 3324 233.1 X-12668 0.4174 2.33E−05 2318 246.1 3-phenylpropionate 0.4068 3.91E−05 2830 149.1 (hydrocinnamate) mannose 0.4027 4.74E−05 1753.5 203.9 indoleacrylate 0.3979 5.94E−05 2529 186.1 3-(4-hydroxyphenyl) 0.3873 9.69E−05 1727.9 179.1 propionate X-17147 0.3873  9.7E−05 4830 187.2 3-indoxyl sulfate 0.3822 0.0001 2258 212 anserine 0.3713 0.0002 1126 239.2 arginine 0.3650 0.0003 728 173.2 X-17010 0.3642 0.0003 3169.5 189 cytidine 0.3603 0.0003 1065 244 X-11549 0.3595 0.0003 5093 339.3 5-aminovalerate 0.3585 0.0003 860 118.1 cystine −0.3583 0.0003 2015.3 145.9 prolylhydroxyproline −0.3570 0.0004 960 229.2 arabonate −0.3561 0.0004 1736 292.1 X-12212 0.3490 0.0005 3607 229.1 indolepropionate 0.3479 0.0005 1925 202.1 3-hydroxybutyrate 0.3429 0.0006 1203.5 116.9 (BHBA) X-12435 0.3416 0.0007 3174 357.2 2-aminobutyrate 0.3403 0.0007 758 104.1 3-(3-hydroxyphenyl) 0.3396 0.0007 2000 165.1 propionate docosapentaenoate 0.3394 0.0007 5574 329.4 (DPA; 22:5n3) 2-hydroxybutyrate 0.3387 0.0007 1169.4 130.9 (AHB) adrenate (22:4n6) 0.3355 0.0008 5684 331.3 3-methyl-2-oxobutyrate 0.3354 0.0008 1489 115.1 X-17381 0.3348 0.0009 4159.8 293.1 cysteine −0.3321 0.0009 1560.1 218 X-12189 −0.3305 0.0010 1249 273.1 alpha- 0.3294 0.0011 1208 145.1 hydroxyisovalerate isoleucine 0.3292 0.0011 1614 132.1 X-12104 −0.3275 0.0011 1755 271.1 X-17314 0.3261 0.0012 2595 266.2 X-15546 −0.3239 0.0013 1861 271.1 N-palmitoyl taurine 0.3210 0.0014 5556.8 362.4 3-methyl-2- 0.3192 0.0015 2106 129.2 oxovalerate X-18559 0.3192 0.0015 1864.9 254.1 5-methylcytidine 0.3184 0.0016 1388 258 X-14588 −0.3179 0.0016 648 151 kynurenate −0.3147 0.0018 2243 188.1 X-12195 −0.3142 0.0018 3369 333 valine 0.3097 0.0021 1040 118.1 N-acetylalanine −0.3067 0.0024 882 130.1 phenyl- 0.3056 0.0025 3349 352.2 alanyltryptophan X-12672 0.3025 0.0027 1797 256 X-16869 −0.3013 0.0029 5179.8 330.2 alpha- 0.2966 0.0033 1274 102.8 hydroxyisocaproate N-stearoyl taurine 0.2961 0.0034 5785.9 390.3 glycerol 0.2955 0.0035 1311 205 dihomolinoleate 0.2952 0.0035 5722 307.3 (20:2n6) X-11541 0.2941 0.0036 4984 399 stearate (18:0) 0.2933 0.0037 5886 283.4 N-acetylisoleucine 0.2916 0.0039 3370 174.1 phenyllactate (PLA) 0.2912 0.0040 2237 165.1 X-11949 0.2905 0.0041 3830 220.1 palmitate (16:0) 0.2883 0.0044 5619 255.3 X-11643 0.2862 0.0047 3961 291.2 N-acetyl-beta-alanine 0.2859 0.0047 1460 132.1 arabitol −0.2831 0.0052 1687.5 307.1 taurolithocholate 0.2808 0.0056 5328 482.4 X-12216 0.2782 0.0061 1701 228.1 glutamate 0.2756 0.0066 1611.9 246 17-methylstearate 0.2754 0.0066 5987 297.4 X-13695 −0.2752 0.0067 2511 245 X-12236 0.2738 0.0069 1321 245.1 docosapentaenoate 0.2718 0.0074 5625 329.4 (n6 DPA; 22:5n6) sphingomyelin 0.2701 0.0078 2645 311.3 glycerate 0.2689 0.0081 1360.7 189 3-hydroxyisobutyrate 0.2636 0.0095 1205 177.11 oleate (18:1n9) 0.2626 0.0098 1984.4 339.2 X-11378 0.2620 0.0099 5325 445.4

TABLE 7 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound). ID A B 3-dehydrocarnitine 0.54 0.34 X-16009 0.2 0.2 arachidonate (20:4n6) 0.43 4.48 pantothenate (Vitamin B5) 12.83 19.07 X-17612 0.21 0.3 benzoate 0.03 0.03 1,5-anhydroglucitol (1,5-AG) 0.28 0.28 X-11852 0.1 0.1 X-12668 0.13 0.13 3-phenylpropionate (hydrocinnamate) 18.68 0.13 mannose 0.21 1.45 indoleacrylate 0.06 0.06 3-(4-hydroxyphenyl)propionate 14.94 0.38 X-17147 0.07 0.08 3-indoxyl sulfate 0.01 0.01 anserine 4.31 14.13 arginine 1 0.63 X-17010 0.68 2.51 cytidine 6.36 9.3 X-11549 0.13 0.13 5-aminovalerate 3.41 1.61 cystine 0.37 0.37 prolylhydroxyproline 1.19 0.53 arabonate 4.02 1.03 X-12212 0.21 0.21 indolepropionate 0.07 0.07 3-hydroxybutyrate (BHBA) 0.18 0.34 X-12435 0.36 0.36 2-aminobutyrate 1.58 0.83 3-(3-hydroxyphenyl)propionate 0.1 0.1 docosapentaenoate (DPA; 22:5n3) 0.14 4.84 2-hydroxybutyrate (AHB) 0.28 0.1 adrenate (22:4n6) 0.82 5.31 3-methyl-2-oxobutyrate 0.09 0.09 X-17381 0.17 0.17 cysteine 0.21 0.24 X-12189 0.23 0.23 alpha-hydroxyisovalerate 0.32 0.32 isoleucine 0.93 0.7 X-12104 0.51 1.13 X-17314 0.42 0.13 X-15546 0.15 0.15 N-palmitoyl taurine 1 0.94 3-methyl-2-oxovalerate 0.07 0.07 X-18559 0.78 0.78 5-methylcytidine 0.3 0.09 X-14588 0.09 0.09 kynurenate 0.21 0.21 X-12195 0.84 0.08 valine 0.73 0.58 N-acetylalanine 0.71 0.67 phenylalanyltryptophan 0.29 0.29 X-12672 0.72 0.72 X-16869 0.29 0.29 alpha-hydroxyisocaproate 1.72 0.42 N-stearoyl taurine 2.12 1.18 glycerol 2.61 1.85 dihomolinoleate (20:2n6) 0.33 4.06 X-11541 0.36 0.36 stearate (18:0) 0.33 1.26 N-acetylisoleucine 1.2 0.42 phenyllactate (PLA) 4.9 0.4 X-11949 0.28 0.28 palmitate (16:0) 0.4 0.88 X-11643 0.22 0.22 N-acetyl-beta-alanine 0.39 0.39 arabitol 9.47 0.61 taurolithocholate 0.45 2.42 X-12216 0.25 0.25 glutamate 9.9 25.24 17-methylstearate 0.21 0.32 X-13695 0.11 0.11 X-12236 0.1 0.1 docosapentaenoate (n6 DPA; 22:5n6) 0.61 4.77 sphingomyelin 0.03 0.23 glycerate 3.38 9.94 3-hydroxyisobutyrate 0.12 0.12 oleate (18:1n9) 0.34 1 X-11378 0.05 0.11

TABLE 8 25(OH) Vitamin D3 in response to diet A and Diet B. Diet A Diet B Difference Parameter Mean StdDev Mean StdDev Mean StdErr p-value 25(OH) 95.4 7.5 121.5 7.4 −26.1 3.1 0.001 Vitamin D3

TABLE 9 Compounds validated by dietary change. ID Retention Index Mass arachidonate (20:4n6) 5525 303.4 pantothenate (Vitamin B5) 2218 220.1 X-17612 4298.5 567.4 mannose 1753.5 203.9 X-17147 4830 187.2 anserine 1126 239.2 X-17010 3169.5 189 cytidine 1065 244 prolylhydroxyproline 960 229.2 arabonate 1736 292.1 3-hydroxybutyrate (BHBA) 1203.5 116.9 docosapentaenoate (DPA; 22:5n3) 5574 329.4 adrenate (22:4n6) 5684 331.3 X-12195 3369 333 N-acetylalanine 882 130.1 dihomolinoleate (20:2n6) 5722 307.3 stearate (18:0) 5886 283.4 palmitate (16:0) 5619 255.3 arabitol 1687.5 307.1 taurolithocholate 5328 482.4 glutamate 1611.9 246 17-methylstearate 5987 297.4 docosapentaenoate (n6 DPA; 22:5n6) 5625 329.4 sphingomyelin 2645 311.3 glycerate 1360.7 189 oleate (18:1n9) 1984.4 339.2 X-11378 5325 445.4

Example 3 (1,25(OH)₂ Vitamin D3)

Metabolite correlations with 1,25(OH)₂ vitamin D3 were determined based on plasma metabolomics (Table 10). This provided a predictive model of compounds which can influence 1,25 (OH)₂ vitamin D3 either positively or negatively. Feeding different levels of these compounds (diet B vs diet A; Table 11), and noting changes in 1,25(OH)₂ vitamin D3 (Table 12), served as a validation model. The metabolite compositions of the two different diets were determined in order to identify relative levels of specific compounds. Those compounds validated by the model are shown in Table 13).

TABLE 10 Metabolite correlations with 1,25(OH)₂ vitamin D3. Correlations with a P value < 0.01 are reported. Corre- Correlation Retention ID lation P-Value Index Mass arachidonate (20:4n6) 0.4230 1.76E−05 5525 303.4 X-11378 0.4144 2.71E−05 5325 445.4 X-16009 0.3928 7.53E−05 2191 222 X-11949 0.3776 1.49E−04 3830 220.1 pantothenate (Vitamin B5) 0.3683 2.22E−04 2218 220.1 2-aminobutyrate 0.3516 4.44E−04 758 104.1 cysteine-glutathione −0.3512 4.50E−04 821 427.1 disulfide mannose 0.3503 4.68E−04 1753.5 203.9 X-11549 0.3474 5.25E−04 5093 339.3 X-11787 −0.3408 6.79E−04 1126 148.1 glutaroylcarnitine (C5) 0.3321 9.47E−04 1565 276.1 isobutyrylcarnitine (C4) 0.3308 9.96E−04 1941 232.2 2-hydroxybutyrate (AHB) 0.3208 0.001 1169.4 130.9 sphingomyelin 0.3206 0.001 2645 311.3 3-hydroxybutyrate (BHBA) 0.3157 0.002 1203.5 116.9 prolylhydroxyproline −0.3084 0.002 960 229.2 glycerate 0.3057 0.002 1360.7 189 X-18558 0.2930 0.004 1676.8 380.1 X-17629 0.2919 0.004 680.6 351.1 dihomolinolenate 0.2912 0.004 5600 305.4 (20:3n3 or 3n6) X-17010 0.2898 0.004 3169.5 189 X-12189 −0.2896 0.004 1249 273.1 X-16869 −0.2870 0.005 5179.8 330.2 adrenate (22:4n6) 0.2855 0.005 5684 331.3 X-16120 0.2851 0.005 1271.7 164.1 cysteine −0.2798 0.006 1560.1 218 alpha-tocopherol 0.2796 0.006 2305.4 502.5 5-methylcytidine 0.2769 0.006 1388 258 tyrosine −0.2754 0.007 1516 182.1 hydroquinone sulfate 0.2749 0.007 1383 189 X-17502 −0.2744 0.007 5093.4 297.3 X-13835 0.2733 0.007 1014 169.1 caproate (6:0) 0.2727 0.007 2766 115.2 phosphoethanolamine (PE) −0.2704 0.008 1577.3 299.1 2-linoleoyl-GPE* (18:2) −0.2687 0.008 5650 476.4 octanoylcarnitine (C8) 0.2626 0.010 4100 288.3

TABLE 11 Compound levels present in diets A and B. Values represent scaled imputed values (data are scaled such that the median value measured across all samples was set to 1.0 and missing values (if any) were imputed with the observed minimum for that particular compound). ID A B arachidonate (20:4n6) 0.43 4.48 X-11378 0.05 0.11 X-16009 0.2 0.2 X-11949 0.28 0.28 pantothenate (Vitamin B5) 12.83 19.07 2-aminobutyrate 1.58 0.83 cysteine-glutathione disulfide 0.04 0.04 mannose 0.21 1.45 X-11549 0.13 0.13 X-11787 0.04 0.06 glutaroylcarnitine (C5) 0.27 0.27 isobutyrylcarnitine (C4) 0.31 0.82 2-hydroxybutyrate (AHB) 0.28 0.1 sphingomyelin 0.03 0.23 3-hydroxybutyrate (BHBA) 0.18 0.34 prolylhydroxyproline 1.19 0.53 glycerate 3.38 9.94 X-18558 0.55 0.47 X-17629 0.3 0.09 dihomolinolenate (20:3n3 or 3n6) 0.42 5.03 X-17010 0.68 2.51 X-12189 0.23 0.23 X-16869 0.29 0.29 adrenate (22:4n6) 0.82 5.31 X-16120 0.45 0.45 cysteine 0.21 0.24 alpha-tocopherol 0.01 0.03 5-methylcytidine 0.3 0.09 tyrosine 0.82 0.86 hydroquinone sulfate 0.03 0.03 X-17502 0.42 1.15 X-13835 0.4 0.4 caproate (6:0) 0.35 0.49 phosphoethanolamine (PE) 1.68 5.87 2-linoleoyl-GPE* (18:2) 0.56 2.58 octanoylcarnitine (C8) 0.41 0.53

TABLE 12 1, 25(OH)₂ vitamin D3 in response to diet A and Diet B. Diet A Diet B Difference Parameter Mean StdDev Mean StdDev Mean StdErr p-value 1, 25(OH)₂ 128.8 9.0 150.2 7.4 −21.3 5.1 0.001 Vitamin D3

TABLE 13 Compounds validated by dietary change. ID Retention Index Mass arachidonate (20:4n6) 5525 303.4 X-11378 5325 445.4 pantothenate (Vitamin B5) 2218 220.1 mannose 1753.5 203.9 isobutyrylcarnitine (C4) 1941 232.2 sphingomyelin 2645 311.3 3-hydroxybutyrate (BHBA) 1203.5 116.9 prolylhydroxyproline 960 229.2 glycerate 1360.7 189 dihomolinolenate (20:3n3 or 3n6) 5600 305.4 X-17010 3169.5 189 adrenate (22:4n6) 5684 331.3 alpha-tocopherol 2305.4 502.5 caproate (6:0) 2766 115.2 octanoylcarnitine (C8) 4100 288.3

It should be understood that various changes and modifications to the presently embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims. 

1. A pet food composition comprising: protein, carbohydrates, fat, fiber, and at least 6 distinct metabolites including at least 2 distinct metabolites for modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25(OH)₂ vitamin D3 in a companion animal; wherein the metabolite that modulates bone mineral content is selected from the group consisting of creatinine, X-14625, gamma-glutamylleucine, ethanolamine, histidine, 4-acetylphenyl sulfate, phosphoethanolamine (PE), X-14352, pseudouridine, myo-inositol, X-15375, X-17299, 1-methylguanosine, xylitol, X-11787, pyroglutamine, and mixtures thereof; wherein the metabolite that modulates 25(OH) vitamin D3 is selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), X-17612, mannose, X-17147, anserine, X-17010, cytidine, prolylhydroxyproline, arabonate, 3-hydroxybutyrate (BHBA), docosapentaenoate (DPA; 22:5n3), adrenate (22:4n6), X-12195, N-acetylalanine, dihomolinoleate (20:2n6), stearate (18:0), palmitate (16:0), arabitol, taurolithocholate, glutamate, 17-methylstearate, docosapentaenoate (n6 DPA; 22:5n6), sphingomyelin, glycerate, oleate (18:1n9), X-11378, and mixtures thereof; wherein the metabolite that modulates 1,25(OH)₂ vitamin D3 is selected from the group consisting of arachidonate (20:4n6), X-11378, pantothenate (Vitamin B5), mannose, isobutyrylcarnitine (C4), sphingomyelin, 3-hydroxybutyrate (BHBA), prolylhydroxyproline, glycerate, dihomolinolenate (20:3n3 or 3n6), X-17010, adrenate (22:4n6), alpha-tocopherol, caproate (6:0), octanoylcarnitine (C8), and mixtures thereof; and wherein the pet food composition provides at least a 5% increase or at least a 5% decrease in the amount of at least one of bone mineral content, 25(OH) vitamin D3, or 1,25 (OH)₂ vitamin D3 in the companion animal upon administration of the pet food to the companion animal.
 2. The pet food composition of claim 1, wherein the composition comprises at least 9 distinct metabolites, including at least 3 distinct metabolites for modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25 (OH)₂ vitamin D3 in a companion animal.
 3. The pet food composition of claim 1, wherein the composition comprises at least 12 distinct metabolites, including at least 4 distinct metabolites for modulating each one of bone mineral content, 25(OH) vitamin D3, and 1,25 (OH)₂ vitamin D3 in a companion animal.
 4. The pet food composition of claim 1, wherein the metabolite modulates bone mineral content and is selected from the group consisting of creatinine, gamma-glutamylleucine, ethanolamine, histidine, 4-acetylphenyl sulfate, phosphoethanolamine (PE), pseudouridine, myo-inositol, 1-methylguanosine, xylitol, pyroglutamine, and mixtures thereof.
 5. The pet food composition of claim 1, wherein the metabolite modulates 25(OH) vitamin D3 and is selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), mannose, anserine, cytidine, prolylhydroxyproline, arabonate, 3-hydroxybutyrate (BHBA), docosapentaenoate (DPA; 22:5n3), adrenate (22:4n6), N-acetylalanine, dihomolinoleate (20:2n6), stearate (18:0), palmitate (16:0), arabitol, taurolithocholate, glutamate, 17-methylstearate, docosapentaenoate (n6 DPA; 22:5n6), sphingomyelin, glycerate, oleate (18:1n9), and mixtures thereof.
 6. The pet food composition of claim 1, wherein the metabolite modulates 1,25(OH)2 vitamin D3 and is selected from the group consisting of arachidonate (20:4n6), pantothenate (Vitamin B5), mannose, isobutyrylcamitine (C4), sphingomyelin, 3-hydroxybutyrate (BHBA), prolylhydroxyproline, glycerate, dihomolinolenate (20:3n3 or 3n6), adrenate (22:4n6), alpha-tocopherol, caproate (6:0), octanoylcarnitine (C8), and mixtures thereof.
 7. The pet food composition of claim 1, wherein the pet food composition provides at least a 10% increase in at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal.
 8. The pet food composition of claim 1, wherein the pet food composition provides at least a 10% decrease in at least one of bone mineral content, 25(OH) vitamin D3, or 1,25(OH)₂ vitamin D3 in the companion animal. 